Immunoprecipitation and Immune Complex Kinase Assay

- from the Bart’s Lab. Click here for full protocol link. 

1) In the cold room, wash cells with cold “Tris” (the same stuff used for TC).

Spin down the cells if you are working with suspension cells in a low speed swinging bucket centrifuge, resuspend in approximately 1 ml Tris and spin at 2K in a microfuge.

Wash on plate if adherent cells by aspirating medium, adding 2 to 10 ml Tris, and aspirating again.

Chromatin Immunoprecipitation Cloning Protocol

- from the Farnham lab. Click here for full protocol link.

Day 1

1.  Add formaldehyde directly to tissue culture media to a final concentration of 1%.  We generally use 1 x 107 cells per antibody per timepoint.  For cloning, multiple IPs are performed and pooled at later steps therefore we generally start with 1 x 108 cells.  Incubate adherent cells on a shaking platform and suspension cells on a stir plate for 10 minutes at room temperature. 

2.  Stop the crosslinking reaction by adding glycine to a final concentration of 0.125 M.  Continue to rock or spin at room temp for 5 minutes.

Immunoprecipitation Protocol for Labeled Cells.

- from An Zhou, Johns Hopkins, 1995. Click here for full protocol. 

1. To your sample, add Super E, protease inhibitors, 1 mM cold Met, antibody.

TMT extract or medium - dilute with Super E to convenient volume; add protease inhibitors, cold Met and antibody.

Some antibodies work only after the antigen has been denatured (e.g. Ab 877, Ab 471): to 50 ul of sample in TMT, add 5 µl 10% SDS and incubate at 100 ^oC for 5 min; allow to cool to room temperature for 5 min; add IN THIS ORDER: 25 µl 15% NP-40, 180 µl Super E, 3 µl PMSF, 3 µl inhibitor mix, 10 µl antibody. Super E: 50 mm phosphate, 1% Triton.

Immunoprecipitation Method

Find a full immunoprecipitation protocol from the Hancock Lab. Click here for the full protocol link.

METHODS:

Chromatin Immunoprecipitation from Stem Cells

Click here to link to the video

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type .

0:11 Introduction11

0:27 Formaldehyde Crosslinking Cells27

2:22 Preparing Magnetic Beads142

4:13 Cell Sonication253

5:58 Chromatin Immunoprecipitation, Wash, and Elution358

8:53 Crosslink Reversal and DNA Purification533

9:58 Conclusion

Immunoprecipitation Protocol Details

Find a link to immunoprecipitation protocol with details and specifics, reagents and equipment.

Full protocol link.

Immunoprecipitation Protocol

- from eBioscience (click here for full protocol).

Immunoprecipitation involves the interaction between a protein and its specific antibody, the separation of these immune complexes with Protein G or Protein A, and the subsequent analysis by SDS-PAGE. This technique provides a rapid and simple means to separate a specific protein from whole cell lysates or culture supernatants.

• 1. Sample preparation
  2. Preclearing
  3. Antibody incubation/formation of antibody-antigen complexes
  4. Precipitation
  5. Analysis by SDS-PAGE

Cross Linking and Immunoprecipitation Protocol

- from Rockefeller Click here for link to full protocol.

General method for UV cross-linking of tissue/cell lines

Immunoprecipitation

CIP Treatment

3’ RNA Linker Ligation

PNK Treatment

SDS-PAGE and nitrocellulose transfer

RNA Isolation and Purification

Dephosphorylation

3’ RNA linker ligation